The Marine Hachery: The hatching of Mugil Cephalus

The Marine Hachery: The hatching of Mugil Cephalus: The hatching of Mugil Cephalus     The first injection : - HCG by a rate of 10,000 IU and attend as follows : Add 1 ml of sali...

                                 

OUTDOOR ALGAE

· 1.Sea water is filtered throw a 30 micron
· 2. Sea water is chlorinated by 75 ppm chlorine 5% for
· 24 hours
·2. Dechlorination with addition 75 ppm sodium
· thiosulphate
· 3.Nutrients can then be added according to the table


NUTRUIENTS FOR OUTDOORALGAE CULTURES

Compound                                                Amount

Amonium sulphate                                                        100 grams

Monopotassium phosphate                                            30 grams

Urea                                                                                   5 grams

Fe-EDTA                                                                        15 grams

Trace mineral mix                                                              4grams

Inoculation from the algae room as follow


150 L - Cylinder
1000 L - Tank
10000 L - Tank
Initial stocking density should be 3 to 5 million cells per ml .Harvest when density 20 to 25 million cells per ml
· Culture tank should be scabbed clean and rinsed between each use. DO NOT need to be chlorinated .Chlorination is not recommended because any residual chlorine can have detrimental effect on the rotifers and fish larvae


SOURCES OF CONTAMINATION
1. Air supply system
2 Water supply system
3. Culture tank
4. Dirty hands
5. Glassware
6. Mouth
7. Started culture
8. Air stone
9. Rain
10.Water source

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CONTAMINATION
Several types of non-algae contamination occur in micro algae culture
1. CILIATE:
* Paramecium
* Colpoda
* Euplotes

2-BCTERIA

3-NEMATODES

4- FUNGI

5-ROTIFERS

The hatching of Mugil Cephalus

The hatching of Mugil Cephalus   

The first injection: -
HCG by a rate of 10,000 IU and attend as follows:
Add 1 ml of saline solution 0.9 mg to an explosive hormone and is shaken and the withdrawal of 1 ml and injected by the female in fish muscle in the dorsal fin down the right side.
The purpose of this injection is that it increases the secretion of the hormone Gonadotropin Harmone (GTH)
And alert to dismantle the ovarian eggs
Second injection: -
24 hours after the first injection are injected into the mother at 200 micrograms of LHRH-a and equipped as follows:
Add 2 ml of saline solution 0.9 mg to 1 mg bottle hormone (1000 micrograms)
The amount of hormone necessary for fish 1 kg as follows:
Required dose = weight of fish in kg × 200/1000 × 2/1 = 0.4 mm / kg fish
The purpose of this hormone has an effect 50 times the capacity of the hormone
Gonadotropin A eleasing Hormone (GnRH) ovulation, it happens at a temperature of 23 to 25 after 16 to 20 hours
Placed 2 males and one female n the spawning tank

3- culture of rotifers
Saltwater Rotifers  : Brachionus plicatilis (L Type)
                                  Brachionus rotundiformis (S Type)

rotifers require conditions: 
      Temperature: 22 - 30° C
                        pH: 7.5 - 8.5
 

Rotifers be fed with microalgaes (Nannochloropsis,Tetraselmis, Isochrysis) , SELCO, SUPER SELCO  and Bakers' yeast


The three - day - cycle (72 hours )

1- place 250-l of microalgae into a clean 1000 L tank . Inoculat 25 million rotifers  ( initial rotifers    density is 100 / ml )

2- After 24 hours , add add an addition 250 - l of algae culture

3- After another 24 hours , add 500 - l more algae culture 

4- After another 24 hours , harvest the tank , the rotifers density become 200 : 250 / ml )

2 . INDOOR ALGAE STOCK CULTURES

INDOOR ALGAE STOCK CULTURES

The indoor algae room is separated from the rest of hatchery in order to maintain a sterile environment and minimize the opportunity of contamination by rotifers, bacteria, protozoa and other algae. It is air-conditioned at 20-25oC to maintain cool temperatures. Twenty –four hour illumination (2500-5000 lux) supplied by fluorescent lamps throughout the room provides the light needed for growing monocultures of N.oculata Aeration is supplied by PVC air lines with enough adjustable air valves for each culture, indoor algae cultures consist of five- phases: stock culture in test tubes, 250 Ml Erlenmeyer, 2 L Erlenmeyer flasks, 20 L carboys, and 100 L fiberglass cylinders
ASEPTIC TECHNIQUES
Ideally, the algae room technician should complete all indoor algal duties prior to starting any other hatchery work. This ensures that contaminating organisms are not introduced into the algae room from outside sources. If other work must be done before starting the indoor algae duties, the technician should shower and change into fresh clothing before entering the algae room. Other personnel are generally prohibited from entering unless absolutely necessary. Supplies and equipment are cleaned before being brought into the algae room.
In starting and maintaining algae culture, all supplies are soaked overnight in detergent, washed and scrubbed well, then rinsed thoroughly to remove any soapy residue. Glassware is acid washed with 10% HCl, rinsed thoroughly and allowed to air dry overnight before use
Culture vessels and seawater medium must be sterilized. Seawater is filtered through 5and 1-micron cartridge filters. Sterilization of culture vessels occurs either by use of an autoclave or by chlorination, glassware and its algal medium is sterilized in an autoclave
The algae room is sterilized weekly by flooding the floor of the room with fresh water and sprinkling it with powdered chlorine. The water is allowed to evaporate over night and/or empty through floor drains.
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CULTURE METHODSIt starts by transferring pure colonies from the Petri dish to the test tubes. After the growth period (From 7 - 10 days) transfer algae from test tubes to the other different stages
** Test tube stock cultureStock cultures are started in 15 ml test tubes equipped with scrow top caps. Cultures are allowed to grow for one month before transferring. A clean test tube culture with no contamination is selected to laterally transfer into four new test tubes.
Clean test tubes are first filled with 10 ml of sea water and f/2 media. They are then sterilled by autoclaving. Caps are loosely scrowed on prior to autoclaving to allow steam to escape during the process. After autoclaving, the test tubes are cooled and caps are tightened.
Inoculation of new tubes is accomplished with sterile, microbiogical techniques.Transfar are carried out a fume hood equipped with a Bunsen burner and an ultraviolet (UV) light which kills most micro-organisms. The UV light should be turned on 15 minutes prior to use and turned off while the technician performs the inoculations under the hood. The surface of the work area should also be wiped clean with alcohol before use.
A harvestable tube and a new tube are uncapped and the lip of each test tube is waved back and forth over the flame of the Bunsen burner several times. Two milliliters of algae from the harvestable test tube are removed with a sterile pipette and transferred into the new tube. The lips of the test tube are flamed again, and the test tubes are loosely recapped. This is repeated to produce as many new test tubes as possible from the original test tube. A new, sterile pipette should be used for each transfer. Inoculated test tubes are placed on the flask shelves. These test tubes are inverted daily to resuspend algae cells which allowed cells to receive more light, thereby increasing growth.

TWO-Liter Flask Cultures
Flasks are assembled with glass rods, a rubber stopper and air line tubing. The (air outlet tube ) which allows air to be released from the flask, is a short piece of glass rod bent at a45-degree angle which extends over the flask lip to minimize contamination.
Before autoclaving, flasks are assembled and filled with 700 ml seawater and media. Aluminum foil is used to loosely cover the top of an assembled flask and is taped at the nick. The flask is autoclaved and then cooled overnight before use. The contents of the autoclave flasks will appear cloudy as a result of precipitation of the nutrients
To start a new flask culture, a single, clean, uncontaminated test tube culture is selected to inoculate
Tow flasks .these first two are allowed to grow for two weeks to a density of 100 million cell / ml .F each flask is then transferred into two other 2 L flask following inoculation, the initial densities are 10 to 20 million cells / ml which increase to 100 to 150 million cell / ml after 7 days. Once the cultures attain these harvest densities, 400 ml from each of the four flask is laterally transferred to two flasks while the remainder of the culture is used to inoculate a 10 L carboy. Once the flask cultures have been established, lateral transfers of flasks should accrue daily with the oldest cleanest culture, still in the log phase, being chosen each day to transfer. It is highly recommended that at least 14 flasks be cultured at the same time. This number is more than necessary, but provides insurance against algae culture crashes.
To laterally transfer one flask into another flask, the foil from a clean autoclaved flask is removed and air line a fixed to the long glass tube. The flask is aerated vigorously for several minutes so its contents are well mixed before inoculating. With the rubber stopper in place, the exterior of a harvestable flask is rinsed with tab water to remove sedimentation from the upper portion of the flask. Tap water is flushed through the air line of the harvestable flask to replace the amount of water evaporated during the culture period. Approximately 400ml of algae from this flask is then used to inoculate the new flask and the remainder is used to inoculate carboy .aeration should be sufficient so that all algae cells are exposed to light
It is very important that laterally transferred flask flask culture be slowly replaced be new stock every three month to reduced the chances of contamination .new batches of flasks should be inoculate with tube cultures. Old batches of flasks are slowly “phased out" and should be used to inoculate carboys, they should not be used for lateral transfers.
**20 Liter carboy culturesThe carboys are filled with 16 L of filtered seawater and chlorinated with liquid bleach for 24 hours, then neutralized with sodium thiosulphate and gentle aeration for at least 48 hours. The aeration is then turned until they are ready to be inoculated. Nutrients are added just prior to adding the inoculums.
The carboys are inoculated at 7 to 10 million cells /ml and reach harvest densities 40 to 70 million cells/ml after seven days.
** 100-L fiberglass CylinderThe Cylinders filled with filtered seawater and chlorinated with liquid bleach for 24 hours, then neutralized with sodium thiosulphate and gentle aeration for at least 24 hours. The aeration is then turned until they are ready to be inoculated. Nutrients are added just prior to adding the inoculum.
The carboys are inoculated at 4 to 6 million cells /ml and reach harvest densities 30 to 40 million cells/ml after seven days. A harvestable Cylinders is then used to feed the larvae and rotifer

** NUTRINT MEDIA
The medium used is the F/2 formulation of GUILLARD 1962
STOCK SOLUTION

1- SODIUM NITRATE (NaNO3)75 g dissolved in 1000 ml D.W

2- SODIUM DIHYDRGEN ORTHOPHOSPHATE (NaH2PO4.H2O)5 g dissolved in 1000 ml D.W

3- SODIUM SILICATE30 g dissolved in 1000 ml D.W

4- EDTA – TRACE METAL5 g Fe-EDTA +
1 ml CoCl2.6H2O
1ml MnCl2.4H2
1ml NaMoO4.2H2O
1 ml ZnSO4.7H2O
1 ml CuSO4.5H2O
5-VITAMINS1 ml THIAMINE HCl
1 ml BIOTINE
1 ml B12
THE MEDIA IS USED WITH ADATION 1 ML FROM EACH (NITRATE, PHOSPHATE, SILICATE, EDTA – TRACE METAL)

NOTES:SODIUM SILICATE is used for ISOCHRYSIS and DITOMES


** HOW TO PRERAR STOCK SOLUTION OF TRACMETALS
TRACE METAL STOCK SOLUTIONCuSO4.5H2O 4.9 g dissolved in 100 ml D.W
ZnSO4.7H2O 2.2 g dissolved in 100 ml D.W
CoCl.6H2O 1.0 g dissolved in 100 ml D.W
MnCl2.4H2O 18.0 g dissolved in 100ml D.W
Na2MoO4.2H2O 0.63 g dissolved in 100 ml D.W

** HOW TO PREPARE THE VITAMINS
VITAMIN STOCK SOLUATION
THIAMINE HCL 0.1 g dissolved in 1000 D.W

BIOTINE 1.0 g dissolved in 1000 ml D.W

B 12 1.0 g dissolved 1000 ml D.W

1. live feeds production

The foundation of larval rearing in marine fish and many other fish species is live feed production.
Commercial dry feeds, while being the first choice based on convenience, are generally not attractive to larvae less than 25 days .It is believed that motion is an important stimulus which elicits early feeding behavior. Providing inanimate dry feed particles is therefore unsuitable until the larvae become more developed
Culture techniques have been developed for a number of live feed organisms, with two of the most common being the rotifer, Brachionus plicatilis and the nauplius stage of Artemia Salina. Both of these organisms are extensively used for sea bream, sea bass and mullet larval rearing. Rotifers, which rang in the size from 100- 200 micron, are provided during the first 21-28 days, while Artemia nauplii (450-500 micron) are provided on day 13 until the fry are harvested.
The nutritional quality of rotifers as a larval fish feed is related to the quality of feed they are provided while being cultured. The most common types of rotifer feeds are microscopic unicellular algae and yeast, as they both contain high levels of highly unsaturated fatty acids (HUFAs). Algae are third type of live feeds culture required for marine fish larval rearing.
Micro algae are use in marine hatcheries as a food source for larvae

TYPES OF MICROALGAE USED IN MARINE HATCHERIES

1. Nannochloropsis Oculata

2. Isochrysis Galbana

3. Tetraselmis Cuecica

** CHRACTERISTICS OF Nannochlopsis Ocualta
1. Belong to Golden Brown Algae
2. No motile algae
3. Small size 2 – 3 Micron
4. High unsaturated fatty acids (HUFAS)
5. Cell count reach 60 million per ml
6. Used as food source for Rotifers, Clams, marine fish larvae and larvae shrimp
7. The optimum conditions of algae growing
TEMP 20 - 22 C0
LIGHT 2500 - 5000 LUX
SATINTY 25 - 27 PPT
PH 8.2 - 8.7

** CHRACTERISTICS OF Isochrysis Galbana
1. Belong to Golden Brown Algae
2. Motile cell with 2 flagella
3. Golden in color and has a red eyespot
4. Used as food sours for Rotifers, Clams, marine fish larvae and larvae shrimp
5. Cell count reach 15 million per ml
6. The same conditions but temperature need higher than
Nannochloropsis Oculata

** CHARACTERISTICS OF Tetraselmis suecica
1. Belong to green algae
2. Motile with 4 flagella
3. 9 – 10 MICRON wide, 12 - 14 MICRON long
4. Used as food source for Rotifers, Clams,
5. Cell count reach 4 million per ml*************************************************************